DEGs at different time points of the oestrous cycle in the oviduct during the luteal phase
During the oestrous cycle, ovarian steroid hormones, including oestrogen and progesterone, affect oviductal, morphological, and functional responses . Thus, it is important to understand the changes in molecular mechanisms and functions of the oviduct during the oestrous cycle. In this study, the molecular mechanisms regulating dynamic changes in the transcriptome at different time points of the oestrous cycle were identified. As shown in the MDS plot, during the luteal phase (Days 6, 9, 12), samples were grouped together by transcriptome expression on each day and showed similar expression patterns (Fig. 1B). The numbers of DEGs on Days 6, 9, and 12 were 1,405, 1,879, and 2,343, respectively, indicating that the number of significant DEGs gradually increased during luteal phase. Among KEGG pathways enriched in the luteal phase (Fig. 3), calcium signalling pathway plays an important role in normal oocyte development in the oviduct , while ECM–receptor interaction plays a role in protecting and supporting cells during ovulation [28, 29]. Focal adhesions are involved in receptor binding to the ECM, which is crucial for oocyte fertilization . Oocyte meiosis is regulated by progesterone, which promotes oocyte development until fertilization. The PI3K-Akt signalling pathway regulates cell processes such as metabolism, proliferation, and cell survival .
Cell-to-cell interaction and proliferation in the oviduct
Using k-means clustering, we selected three clusters with similar gene expression patterns based on the KEGG pathway database (Fig. 3). The overall physiological changes through the course of oestrous cycle were identified by integrating and analysing Clusters 1 and 2. In particular, the significantly enriched pathways, including ECM–receptor interaction, PI3K-Akt signalling, protein digestion and absorption, glycosaminoglycan biosynthesis, and proteoglycans in cancer, were remarkably associated with oviductal activities during the oestrous cycle. The PI3K-Akt signalling pathway is related to cell physiology and proliferation  and stimulates follicle development and oocyte growth . The ECM plays an essential role in embryo development and regulates different cellular processes via expression of ECM proteins [32, 33]. Moreover, interactions between the cumulus–oocyte complex and epithelial cells of the oviduct are regulated at the level of the ECM . Through its complexes of elastin, proteoglycans, and glycosaminoglycans, the ECM also causes uterus dilation through collagen fibre degradation during the oestrous cycle . Overall, our results revealed significant roles played by time-dependent transcriptomic changes in cell growth, proliferation, and cell–cell interaction during the oestrous cycle.
Oocyte meiosis and calcium signalling pathway
The expression levels of Cluster 3 genes gradually increased over the luteal phase and rapidly decreased after Day 12 (Fig. 5A). Moreover, oocyte meiosis and calcium signalling pathway were most significantly enriched in Cluster 3 genes on Day 12. Furthermore, similar results appeared in the top terms when listed in an order of significance based on the pathways activated on Day 12 and identified via GSEA (Fig. 6A).
Oocyte meiosis is related to the formation and development of oocytes and is an important process involved in oocyte migration and fertilization, the core functions of the oviduct . IGF1, PGR, ITPR3, CAMK2A, CAMK2B, IP3R, BUB1, and CDC20 were downregulated in the enriched KEGG pathways (Fig. 6B and 6D). Previously, IGF1, PGR, ITPR3, CAMK2B, and PLK1 were shown to be involved in oocyte-related processes. IGF1 plays as an important role during early oestrous cycle, and ovulation fails if IGF1 is not expressed . In vivo, IGF1 may indirectly induce the stimulation of cumulus and/or granulosa cells, resulting in improved oocyte maturation and fertilization . PGR is a progesterone receptor that mediates the effects of progesterone in the oviduct. It plays an important role in the successful release of oocytes from preovulatory follicles  as well as in ciliary beat frequency and oocyte transport . ITPR3 is involved in the initiation and propagation of intracellular Ca2+ signalling during the follicular phase , while CAMK2B and PLK1 encode essential factors involved in Ca2+-induced exit from mitosis . Hence, these genes are involved in successful ovulation and fertilization. Therefore, based on the predominance of downregulated genes on Day 12, we suspect that oestrous cycle-related activities take place at a site other than oviduct and active activities occur in follicular phase, where oocytes present in the oviduct.
KEGG pathway analysis identified calcium signalling as the most enriched pathway. Progesterone, an ovarian steroid hormone secreted in the oviduct, activates calcium channels and increases intracellular calcium levels in sperm to promote their hyperactivity . Reportedly, sperm hyperactivity assists in escaping epithelial folds, allowing sperm to reach oocytes . Among the significant DEGs identified by GSEA, ITPR1 and CAMK1D are involved in calcium signalling pathways and associated with fertilization (Fig. 6C and 6E), ITPR1 modulates intracellular calcium ion concentrations during mammalian fertilization ; CAMK1D also serves as a mediator of calcium-dependent cellular processes . We speculate that the predominance of downregulated genes during the luteal phase (Day 12) may indicate their roles being less relevant to fertilization.
As mentioned earlier, oocyte transport is an essential function of the oviduct. In the oviduct, oestrogen and progesterone regulate oocyte transport, cilia beating, and smooth muscle contraction . Cluster profiling revealed a gene involved in calcium signalling pathway that plays a role in the smooth muscle contraction in the oviduct (Fig. 6E). DEGs related to oocyte transport were identified by functional analysis and GSEA: activation of CHRM3 is associated with muscle contraction as the M3 receptor mediates intracellular calcium , while TNNC1 is involved in calcium contractile events by binding with Ca2+ . Downregulation of these genes on Day 12 and observation of smooth muscle relaxation explains why muscle contraction is inhibited in the luteal phase.